91 results about "Nicking enzyme" patented technology

The present invention provides a method and a kit for detecting a target nucleic acid to be detected by using a melting curve. The method includes designing a first primer, a second primer and a detection probe for the target nucleic acid sequence to be detected, performing PCR amplification in a PCR amplification system containing the first primer, the second primer, the detection probe, the sample to be detected, a nicking enzyme and a DNA polymerase, generating a reporter primer that is complementary to the detection probe to form a double-stranded product, obtaining the melting curve of the double-stranded product, and the melting curve of the double-stranded product is the melting curve corresponding to the target nucleic acid to be detected. The method of the present invention is a non-target-dependent melting curve method, and the melting point (T m value) of each double-stranded body can be calculated in advance, solving the problems of melting curve peak shift and easy misjudgment caused by the easy mutation of the target nucleic acid sequence to be detected.

The invention belongs to the technical field of gene editing, and discloses a gene editing system and application. The invention provides a gene editing system. The gene editing system comprises Cas9 nickase, sgRNA (small guide ribonucleic acid) and annular petRNA. According to the present invention, the target chain cutting mediated reverse pilot editing (Split Reverse Prime Editing, srPE) realizes the precise editing of the non-target chain cutting site upstream sequence, such that the gene editing coverage range is expanded from the specific region only limited to the non-target chain cutting site downstream to the whole genome range, the stable and efficient editing ability is maintained in different cell types, and the target chain cutting mediated reverse pilot editing can be provided for the target chain cutting site. And meanwhile, the off-target risk is reduced. The srPE gene editing technology breaks through the fundamental limitation of the prior art, and provides a more comprehensive and more efficient technical tool for precise medicine and gene therapy.

The invention relates to the technical field of gene editing, in particular to a Cas9 protein mutant and application thereof. According to the present invention, the mutation is introduced at the position far away from the catalytic site to induce the Cas9 protein conformation change so as to obtain the new Cas9 protein mutant, only the Cas9-sgRNA target chain is cut, and the Cas9-sgRNA non-target chain is not cut, such that the new Cas9 protein mutant can be used for constructing the novel base editor, and the high-efficiency base editing can be performed on the mammalian cell genome. The Cas9 protein mutant disclosed by the invention is fused with a mutant TX with DNA binding deletion of 3 '-> 5' exonuclease TREX2, so that the gene editing efficiency of a 3'terminal generated by paired single-nick enzyme cutting can be greatly improved, and the safety of the Cas9 protein mutant is maintained.

Provided is a reverse prime editing system, which is used for achieving site-specific and precise small-fragment DNA editing upstream of a target site. In the reverse prime editing system, a targeted strand nickase (such as D10A-Cas9) is used to cleave a nick on a targeted strand, thereby enabling precise small-fragment DNA base insertion, deletion and substitution upstream of the target site under the action of a reverse transcriptase.

The present disclosure provides CRISPR systems using engineered MAD7 endonucleases, as well as methods, vectors, nucleic acid compositions, and kits thereof. In particular, provided herein are MAD7 nickases, catalytically dead MAD7 enzymes, and hyperactive MAD7 enzymes.

The invention discloses a method for targeted enrichment of polymorphic sites by introducing thio-modified nucleic acid into a single base. According to the invention, the selector contains a segment of primer extension binding region with more than four basic groups, when the selector is extended by taking a target DNA / RNA as a template, single thio-modified dNTP and another three terminated dNTP / ddNTP, denaturation and renaturation are carried out, a hairpin structure is formed by itself, the selector without thio-modification is digested under the action of T4DNA polymerase, and then the target DNA / RNA is converted into the target DNA / RNA, and the target DNA / RNA is converted into the target DNA / RNA, and then the target DNA / RNA is converted into the target DNA / RNA, and the target DNA / RNA is converted into the target DNA / RNA, and the target DNA / RNA is converted into the target DNA / RNA. And complementing the sequence at the incision to form an intermediate product with a hairpin structure, so as to fulfill the aim of enriching rare mutation sites. The method is suitable for DNA and RNA templates, and the obtained hairpin ring structure intermediate can be subjected to signal amplification in two ways: enzyme-mediated nucleic acid amplification or PCR amplification after linearization of a ring structure.

An artificial nicking enzyme comprising two domains ND1 and ND1n linked via an ND1 linker, wherein ND1 is at least one polypeptide selected from the group consisting of (a) a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 98, as well as variants thereof which are polypeptides comprising an amino acid sequence wherein the amino acids corresponding to the 66th and 83rd amino acids of the amino acid sequence set forth in SEQ ID NO: 98 are aspartic acid, and, ND1n is at least one polypeptide selected from the group consisting of (an) a polypeptide comprising an amino acid sequence which is a substituted amino acid wherein the 66th aspartic acid and / or the 83rd aspartic acid of the amino acid sequence set forth in SEQ ID NO: 98 is substituted with another arbitrary amino acid, as well as variants thereof which are polypeptides comprising an amino acid sequence wherein the amino acid corresponding to the 66th aspartic acid and / or the 83rd aspartic acid of the amino acid sequence set forth in SEQ ID NO: 98 is a substituted amino acid substituted with an arbitrary amino acid other than aspartic acid, the nicking enzyme.

The invention relates to a method for simultaneously amplifying a plurality of target nucleic acid sequences in a sample, said method having the step of: (S300) amplifying the target nucleic acid sequences by means of an isothermal strand displacement amplification process using pairs of flanked primers, a superprimer (10), and at least one first nicking enzyme. One pair of flanked primers is used for each target nucleic acid sequence to be amplified, and the pair of flanked primers is specific to the associated target nucleic acid sequence, each pair of flanked primers having a flanked forward primer and a flanked reverse primer; each flanked forward primer has a target-specific portion at the 3' end thereof and a a non-specific flank portion at the 5' end thereof, the target-specific portion being complementary to a portion of the associated target nucleic acid sequence; each flanked reverse primer has a target-specific portion at the 3' end thereof and a non-specific flank portion at the 5' end thereof, said target-specific portion being complementary to a portion of the associated target nucleic acid sequence; and the superprimer (10) has a binding portion (12) at the 3' end thereof and a recognition portion (14) at the 5' end thereof, said recognition portion (14) having a recognition sequence for the first nicking enzyme.

Provided herein are gene editing systems and compositions directed to effectuate in vivo edits in the LPA gene. Treatment or prevention of cardiovascular disease through disruption of the production of apo(a) through genetic editing and the reduction of the blood lipoprotein(a) [Lp(a)] concentration is disclosed herein. Disclosed are nickase-based gene editing systems designed to effectuate the installation of insertions and / or deletions (indel variants) and / or non-synonymous variants in the coding sequence of LPA. The nickase-based gene editing systems generally comprise one or more mRNAs that encode one or more nickases and a plurality of guide oligonucleotides (e.g., gRNAs) and may be delivered in vivo to a mammalian subject in need thereof via a suitable delivery system, such as lipid nanoparticles (LNPs) (with or without GalNAc targeting moieties) intravenously, or otherwise, administered to a patient as potentially a once-and-done therapeutic. The manufacturing, use, and formulation of the gene editing systems and compositions are also disclosed.

This invention pertains to fusion protein mutants comprising a Prime Editing enzyme having a first amino acid sequence and a second amino acid sequence, wherein the first amino acid sequence comprises a SpCas9 H840A nickase mutant protein of SEQ ID NO:152 and the second amino acid sequence comprises a Moloney Murine Leukemia Virus reverse transcriptase protein mutant (MMLV RTase mutant), wherein the fusion protein mutant displays at least the equivalent or greater activity of a reference Prime Editing enzyme in genome editing.

Provided are systems for strand-specific editing of DNA, including mitochondrial DNA in humans. The systems provided herein comprise a nickase and a single-stranded (ss) DNA deaminase each, or together, associated with a double-stranded DNA binding polypeptide such that DNA editing occurs in an editing region. Also provided herein are components of the systems for editing DNA taught herein, and methods of use thereof.

A modified pegRNA Cas9 nickase system that maintains potent on-target editing activity but has reduced off-target editing activity relative to wild-type Cas, and methods of production and use.

This invention discloses a method for producing single-stranded DNA using Cas nickase. This method solves the problem of limitations on the enzyme cleavage sites when using nicking endonucleases, and improves the nicking efficiency of plasmids by performing multiple nicking using multiple gRNAs in a single enzyme cleavage system, while also effectively improving the efficiency of subsequent digestion by exonucleases.

The present application relates to the field of biotechnology, and particularly relates to a method for identifying a cleavage site of a nucleic acid nicking enzyme. The present application discloses a method for identifying a cleavage site of a nicking enzyme, which comprises the following steps: obtaining nucleic acid sequence data before and after enzyme cleavage by using mNGS sequencing, analyzing second-generation sequencing data by using Bowite2 and samtools software, and obtaining the cleavage site of the nicking enzyme according to single-base sequencing depth and analysis. The present application does not need to determine the sequence of the nucleic acid to be cleaved, has short time consumption and low budget, and has a good application prospect.

The invention discloses a Cas12 nuclease mutant, and compared with wild type Cas12 nuclease, the Cas12 nuclease mutant comprises any one or more of the following mutation structural domains: aspartic acid in a highly conservative amino acid sequence of a RuvC nuclease structural domain 1 is mutated into amino acid with hydroxyl or sulfydryl or amide, and aspartic acid in a highly conservative amino acid sequence of a RuvC nuclease structural domain 2 is mutated into amino acid with sulfydryl or amide; arginine in a highly conservative amino acid sequence of the NUC structural domain is mutated into amino acid with a short side chain or a special structure; aspartic acid in a highly conservative amino acid sequence of the RuvC III structural domain is mutated into amino acid with hydroxyl or sulfydryl or amide or with extremely short side chains; the Cas12 nuclease mutant has the activity of a Cas12 nickase. A gene editing system constructed by utilizing the Cas12 nuclease mutant disclosed by the invention can be used for carrying out precise editing on target genes in bacteria and cells.

This application belongs to the field of biotechnology and relates to an efficient and specific editing system, method, and application for A-G base substitution. The fusion protein described in this application comprises, from the N-terminus to the C-terminus, a first SaCas9 nickase fragment, a chimeric deaminase fragment, and a second SaCas9 nickase fragment. The deaminase is selected from adenosine deaminase or a variant thereof, specifically ecTadA8e. The amino acid sequence of ecTadA8e is shown in SEQ ID NO. 20, or has more than 80% sequence identity with the amino acid sequence shown in SEQ ID NO. 20, and possesses the function or activity of ecTadA8e. The fusion protein provided in this application, combined with the corresponding guide RNA, can efficiently and specifically replace the base A with G at the target site, providing an effective tool for repairing pathogenic mutations, studying gene function, and improving cell function, and has promising application prospects.

Disclosures herein are directed to compositions comprising single guide RNA (sgRNA) and fusion proteins comprising a Cas9 nickase and deaminase designed for a CRISPR-Cas9 system and method of using thereof for preventing, ameliorating or treating one or more cardiomyopathies.

This invention discloses a method and kit for detecting RNA using a three-stage amplification melting curve based on isothermal self-circulation. The method includes designing a first primer, a second primer, and a circular amplification probe targeting the target nucleic acid sequence. The first primer consists of an RNA polymerase promoter sequence and a target nucleic acid-specific binding sequence, sequentially from the 5' end to the 3' end. The circular amplification probe is a circular DNA segment whose sequence consists of three parts: the first part completely contains a continuous sequence composed of the tag sequence and the nicking enzyme-specific recognition sequence from the second primer; the second part is a self-cleaving DNase sequence. In the technical solution used in this invention, a cascaded nucleic acid amplification system is constructed, achieving exponential signal amplification of the target nucleic acid through the synergistic effect of rolling circle amplification and RNA polymerase-mediated transcriptional amplification.

The invention provides an exonuclease gene editor and a gene editing system and application thereof, and particularly provides an exonuclease editor formed by fusing exonuclease and Cas9 or nicking enzyme, the exonuclease editor has very good gene editing activity, and compared with Cas9 nuclease, the exonuclease editor can remarkably improve accurate gene editing efficiency. The exonuclease editor provided by the invention can also be used for accurately deleting long-fragment genes.

Disclosed herein include methods, compositions, and kits suitable for use in gene editing. RT editing systems are provided herein comprising a fusion protein comprising a Cas9 nickase and a reverse transcriptase, and a template armed guide RNA (tagRNA) comprising from 5′ to 3′ a spacer sequence, a scaffold sequence, an editing template and a flap binding sequence. In some embodiments, the RT editing system further comprises an enhancer guide RNA (egRNA).

A chemically modified polynucleotide encoding a fusion protein, wherein the chemically modified polynucleotide comprises a sequence of SEQ ID NO: 701 is claimed wherein the fusion comprises a Cas9 nickase and and deaminase, such as an adenosine deamnase TadA. and which further comprises a protospacer found on a gene encoding Angiopoietin-like 3 protein (ANGPTL3). The chemical modifications may include phosphorothioate linkages and 2'-O-methyl modfied nucleosides. Use of such fusions in treating cardiovascular conditions is further claimed. [Figure 3]

The invention provides sgRNA (small guide ribonucleic acid) and target DNA (deoxyribonucleic acid) for detecting the activity of SpCas9 D10A Nickase and a detection method. A reaction system capable of expressing the function and a quantitative detection method (IEX-HPLC method) are established by simulating the enzyme activity function of a base editing enzyme incision, so that the requirements on experimental animals and cells are remarkably reduced, the detection period is shortened, and the 3R (reduction, optimization and substitution) principle of modern scientific research is met; meanwhile, the invention also provides an efficient and practical analysis method for the quality characteristic analysis work of the base editing enzyme, the convenience of quality detection in gene editing product production and research is improved, and the method has a large-scale commercial application prospect.

Provided herein are Cas nucleases (e.g., endonucleases, nickases, etc.) (and functional fragments, functional variants, and domains thereof), nucleic acid molecules encoding the same, and systems comprising the same. The disclosure further relates to methods of utilizing the Cas nucleases (e.g., endonucleases, nickases, etc.) (or nucleic acid molecules encoding the same), including, e.g., in methods of editing a nucleic acid molecule (e.g., a gene) and methods of treating diseases (e.g., genetic diseases).

The invention relates to the technical field of agriculture, in particular to an efficient ABE base editing tool based on Cas12i3 and application of the efficient ABE base editing tool. The Cas12i3-based efficient ABE base editing tool provided by the invention comprises a protein expression cassette and an RNA (Ribonucleic Acid) expression cassette, the protein expression cassette comprises a promoter, a 2 * BP NLS nuclear localization signal fragment, a 2 * adenine deaminase TadA8e, a connecting peptide, a Cas12i3 notch enzyme fragment and a terminator, the RNA expression cassette comprises a promoter, a crRNA sequence and a terminator; wherein the crRNA sequence is composed of a DR sequence and a target specific spacer sequence. The tool realizes efficient and specific single base substitution of A-to-G in a rice genome by utilizing the targeted binding characteristic of Cas12i3 and the efficient deamination activity of 2 * TadA8e, has the advantages of high editing efficiency, low off-target rate, wide application range and the like, provides an efficient and accurate technical means for plant functional genome research and molecular breeding, and has a wide application prospect. The creation of excellent rice varieties can be accelerated.

The present invention relates to the field of genomic engineering. In particular, a chimeric prime editing (cPE) system is disclosed comprising elements including, but not limited to a Cas9 nickase (nCas9) / high fidelity nucleotide polymerase (HFNTPol) RNA, one or more single guide RNAs (sgRNAs), and a chimeric prime editor template oligonucleotide (cpetODN) comprising a deoxyribonucleic acid nucleotide polymerase template (NPT) and a primer binding site. For example, the sgRNA and the cpetODN are ligated into a single oligonucleotide. Alternatively, the sgRNA and the cpetODN are free and independent molecules (e.g., modular). This cPE system results in precise and efficient genome editing in cells and in adult mouse liver which is advantageous over conventional sgRNA prime editor fusion constructs. This flexible and modular system is an improvement in the art to obtain precise genome editing.

Disclosed herein include methods, compositions, and kits suitable for use in gene editing. RT editing systems are provided herein comprising a fusion protein comprising a Cas9 nickase and a reverse transcriptase, and a template armed guide RNA (tagRNA) comprising from 5' to 3' a spacer sequence, a scaffold sequence, an editing template and a flap binding sequence. In some embodiments, the RT editing system further comprises an enhancer guide RNA (egRNA).

The application belongs to the technical field of biological medicine, and discloses a gene leading editing system, which comprises a carrier, the carrier contains a gene coding pegRNA and a gene coding fusion protein, the pegRNA comprises sgRNA, reverse transcription template RTT, primary binding site PBS and RNA stem loop structure, and the fusion protein is a fusion protein of Cas9 nickase or a variant thereof and reverse transcriptase. The application develops sPEs by adding an RNA neck ring structure to the 3' end of the pegRNA, develops tPEs by allowing the 3' end of the pegRNA to bind to Cas9, so as to improve the stability of the Cas9 / pegRNA complex, and further improve the editing efficiency of different genomic sites in different cells.

The invention discloses a rolling circle amplification method and application of the rolling circle amplification method in improvement of nanopore sequencing accuracy. In the construction process of the nanopore sequencing library, two ends of a to-be-detected target linear double-stranded DNA sample are connected with two joints with nick enzyme recognition sites, the joints are digested through the nick enzyme to generate two cohesive ends which are complementarily paired, and the two cohesive ends are connected, so that the target linear double-stranded DNA sample is obtained. According to the method, self-cyclization of a to-be-detected sample is realized to obtain a double-stranded circular DNA as a template for rolling circle amplification, a forward primer and a reverse primer are designed for the template to carry out rolling circle amplification reaction to obtain a nanopore sequencing library, and experiments prove that the nanopore sequencing accuracy can be improved to 99.9% by the method disclosed by the invention; and a longer DNA fragment product can be generated by utilizing rolling circle amplification, and the DNA fragment product is used for nanopore sequencing and the yield of a nanopore sequencing library is improved.

In one aspect, the disclosure relates to a system for site-specific modification of a double­stranded target DNA sequence, the system including at least: a fusion protein comprising an RNA-binding nickase protein and a DNA-dependent DNA polymerase, and a chimeric guide nucleic acid sequence comprising a guide RNA sequence that interacts with the RNA-binding nickase protein and a single-stranded DNA template, wherein the single-stranded DNA template comprises a modified sequence for insertion into a second strand of the double-stranded target DNA sequence and a primer binding site. Also disclosed is a method for site-specific modification of a double-stranded target DNA sequence in a cell. In another aspect, the present disclosure is directed to a fusion protein including an RNA-binding nickase protein and a DNA-dependent DNA polymerase, wherein one or both of the nicakse protein and the DNA polymerase can include one or more mutations to optimize function and prevent off-target effects.